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  • 93
    Agilent technologies bioanalyzer dna high sensitivity chip
    Small RNA library analysis. ( A – C ) HL small RNA library purification and validation. ( A ) Representative samples of amplified cDNA prepared from HL-RNA were chromatographed on a 5% PAGE gel. The first lane in each panel is a marker composed of three dsDNA fragments of 145, 160, and 500 bp. The region between 145 and 160 bps, corresponding to adapter-ligated constructs derived from miRNA, was excised from the gel ( B ), and purified. The libraries prepared using these purified constructs were validated by analysis with Agilent <t>Bioanalyzer</t> High Sensitivity <t>DNA</t> Chip. ( C ) A representative electropherogram of a purified library. The peak at ~150 bp indicates the presence of cDNA from HL-miRNA. The peaks other than the ones labeled as miRNAs are the lower and upper markers used by the Bioanalyzer system to determine the size and quantity of the library peak. ( D , E ) Length distribution and annotation of small RNA sequencing reads from HL and BT. ( D ) Sequencing reads from all HL or BT miRNA samples were pooled for the purpose of length distribution and annotation analyses. Length distributions by abundance of sequencing reads from BT (clear bars) or HL (filled bars) are shown. Only reads 18–28 nucleotides in size which map uniquely to the UCSC Genome Browser dm6 (NCBI genome/47) Drosophila melanogaster genome are shown. ( E ) Distribution of RNA biotypes represented as the percentages of reads mapping to the indicated small RNAs. Clear and filled bars represent BT and HL samples, respectively. “Other” represents other Drosophila sncRNAs.
    Bioanalyzer Dna High Sensitivity Chip, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies bioanalyzer 2100 dna high sensitivity chip
    Small RNA library analysis. ( A – C ) HL small RNA library purification and validation. ( A ) Representative samples of amplified cDNA prepared from HL-RNA were chromatographed on a 5% PAGE gel. The first lane in each panel is a marker composed of three dsDNA fragments of 145, 160, and 500 bp. The region between 145 and 160 bps, corresponding to adapter-ligated constructs derived from miRNA, was excised from the gel ( B ), and purified. The libraries prepared using these purified constructs were validated by analysis with Agilent <t>Bioanalyzer</t> High Sensitivity <t>DNA</t> Chip. ( C ) A representative electropherogram of a purified library. The peak at ~150 bp indicates the presence of cDNA from HL-miRNA. The peaks other than the ones labeled as miRNAs are the lower and upper markers used by the Bioanalyzer system to determine the size and quantity of the library peak. ( D , E ) Length distribution and annotation of small RNA sequencing reads from HL and BT. ( D ) Sequencing reads from all HL or BT miRNA samples were pooled for the purpose of length distribution and annotation analyses. Length distributions by abundance of sequencing reads from BT (clear bars) or HL (filled bars) are shown. Only reads 18–28 nucleotides in size which map uniquely to the UCSC Genome Browser dm6 (NCBI genome/47) Drosophila melanogaster genome are shown. ( E ) Distribution of RNA biotypes represented as the percentages of reads mapping to the indicated small RNAs. Clear and filled bars represent BT and HL samples, respectively. “Other” represents other Drosophila sncRNAs.
    Bioanalyzer 2100 Dna High Sensitivity Chip, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies bioanalyzer dna high sensitivity chips
    Small RNA library analysis. ( A – C ) HL small RNA library purification and validation. ( A ) Representative samples of amplified cDNA prepared from HL-RNA were chromatographed on a 5% PAGE gel. The first lane in each panel is a marker composed of three dsDNA fragments of 145, 160, and 500 bp. The region between 145 and 160 bps, corresponding to adapter-ligated constructs derived from miRNA, was excised from the gel ( B ), and purified. The libraries prepared using these purified constructs were validated by analysis with Agilent <t>Bioanalyzer</t> High Sensitivity <t>DNA</t> Chip. ( C ) A representative electropherogram of a purified library. The peak at ~150 bp indicates the presence of cDNA from HL-miRNA. The peaks other than the ones labeled as miRNAs are the lower and upper markers used by the Bioanalyzer system to determine the size and quantity of the library peak. ( D , E ) Length distribution and annotation of small RNA sequencing reads from HL and BT. ( D ) Sequencing reads from all HL or BT miRNA samples were pooled for the purpose of length distribution and annotation analyses. Length distributions by abundance of sequencing reads from BT (clear bars) or HL (filled bars) are shown. Only reads 18–28 nucleotides in size which map uniquely to the UCSC Genome Browser dm6 (NCBI genome/47) Drosophila melanogaster genome are shown. ( E ) Distribution of RNA biotypes represented as the percentages of reads mapping to the indicated small RNAs. Clear and filled bars represent BT and HL samples, respectively. “Other” represents other Drosophila sncRNAs.
    Bioanalyzer Dna High Sensitivity Chips, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies bioanalyzer 2100 dna high sensitivity chips
    Standards for MspI digestion and progressive PCR. A) MspI digestion of human genomic <t>DNA</t> isolated from human post-mortem brain tissues. DNA (200 ng) was digested by MspI and run on a 4–20% precast polyacrylamide gel and stained with EtBr. Arrows show three satellite DNA bands characteristic of this enzymatic digestion. B) Agilent 2100 Bioanalyzer chromatogram of MspI digested genomic DNA. C) <t>Bioanalyzer</t> 2100 image of a single library from an MspI digested DNA sample. Notice that the satellite bands (indicated by arrows) are still visible on the Bioanalyzer image. D) Progressive PCR amplification combined with limited PCR extension time allows for size selection and amplification of six bisulfite converted libraries (Lanes 1–5 are distinct RRBS libraries; lane 6 (‘C’) is a negative control). After different progressive PCR cycles (18X, 22X, 24X, or 26X – the same libraries are shown for each cycle number) band intensity increases as cycle number increases. Arrows indicate the three satellite DNA bands that are still visible in these libraries.
    Bioanalyzer 2100 Dna High Sensitivity Chips, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Agilent technologies dna high sensitivity chip
    <t>DNA</t> methylation levels inversely correlate with expression levels of Esrp2 and Esrp2-as . ( a ) Upper : genomic organization of Esrp2 (dark blue) and Esrp2-as variants 1-4 (v1-4, light blue) and the CGI overlapping the Esrp2 TSS (green). Positions of EPITYPER Amplicons A1-A14 are indicated by horizontal black bars, covering the DMR (pink), the CGI, and CGI shores on both sides. Lower : <t>MCIp-seq</t> detection of methylated DNA fragments in tumors (red) and normal WT mammary glands (blue) of animals at 20 and 24 weeks of age. Each lane represents average reads obtained for three individual samples. ( b , c ) Heatmap of DNA methylation levels in tumor samples ( n =11) and normal mammary gland tissue ( n =9) ( b ), or of various murine cell lines ( c ), with each row representing one individual sample and each column one CpG unit comprising of 1 to 4 individual CpG sites. Methylation levels are depicted by a color-coded gradient from 0% (light yellow) to 100% methylation (blue). Gray squares indicate failed measurements. ( d , e ) Correlation between average methylation of amplicons A1-A14 and Esrp2/Esrp2-as expression levels normalized to three reference genes, calculated by Spearman’s rank correlation for tumor/normal tissues ( d ) and cell lines ( e ). * P
    Dna High Sensitivity Chip, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 97/100, based on 920 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agilent technologies bioanalyzer dna high sensitivity kit
    <t>DNA</t> methylation levels inversely correlate with expression levels of Esrp2 and Esrp2-as . ( a ) Upper : genomic organization of Esrp2 (dark blue) and Esrp2-as variants 1-4 (v1-4, light blue) and the CGI overlapping the Esrp2 TSS (green). Positions of EPITYPER Amplicons A1-A14 are indicated by horizontal black bars, covering the DMR (pink), the CGI, and CGI shores on both sides. Lower : <t>MCIp-seq</t> detection of methylated DNA fragments in tumors (red) and normal WT mammary glands (blue) of animals at 20 and 24 weeks of age. Each lane represents average reads obtained for three individual samples. ( b , c ) Heatmap of DNA methylation levels in tumor samples ( n =11) and normal mammary gland tissue ( n =9) ( b ), or of various murine cell lines ( c ), with each row representing one individual sample and each column one CpG unit comprising of 1 to 4 individual CpG sites. Methylation levels are depicted by a color-coded gradient from 0% (light yellow) to 100% methylation (blue). Gray squares indicate failed measurements. ( d , e ) Correlation between average methylation of amplicons A1-A14 and Esrp2/Esrp2-as expression levels normalized to three reference genes, calculated by Spearman’s rank correlation for tumor/normal tissues ( d ) and cell lines ( e ). * P
    Bioanalyzer Dna High Sensitivity Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Agilent technologies bioanalyzer high sensitivity chip
    <t>DNA</t> methylation levels inversely correlate with expression levels of Esrp2 and Esrp2-as . ( a ) Upper : genomic organization of Esrp2 (dark blue) and Esrp2-as variants 1-4 (v1-4, light blue) and the CGI overlapping the Esrp2 TSS (green). Positions of EPITYPER Amplicons A1-A14 are indicated by horizontal black bars, covering the DMR (pink), the CGI, and CGI shores on both sides. Lower : <t>MCIp-seq</t> detection of methylated DNA fragments in tumors (red) and normal WT mammary glands (blue) of animals at 20 and 24 weeks of age. Each lane represents average reads obtained for three individual samples. ( b , c ) Heatmap of DNA methylation levels in tumor samples ( n =11) and normal mammary gland tissue ( n =9) ( b ), or of various murine cell lines ( c ), with each row representing one individual sample and each column one CpG unit comprising of 1 to 4 individual CpG sites. Methylation levels are depicted by a color-coded gradient from 0% (light yellow) to 100% methylation (blue). Gray squares indicate failed measurements. ( d , e ) Correlation between average methylation of amplicons A1-A14 and Esrp2/Esrp2-as expression levels normalized to three reference genes, calculated by Spearman’s rank correlation for tumor/normal tissues ( d ) and cell lines ( e ). * P
    Bioanalyzer High Sensitivity Chip, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 96/100, based on 778 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Agilent technologies bioanalyzer dna high sensitivity chip kit
    L-EVs isolated from plasma of mCRPC patients contain large size <t>DNA</t> with tumour aberrations . (a) DNA quantitation in plasma-derived L-EVs and S-EVs, as well as EV-free DNA obtained from mCRPC patients ( n = 4) indicates that a significant portion of circulating DNA is enclosed in L-EVs. The EV-free DNA was extracted from EV-depleted plasma. (b) Chip-based capillary electrophoresis <t>(Bioanalyzer)</t> showing that L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 3) contain high-quality, large size DNA. (c) EVs were isolated from 1 ml of plasma obtained from mCRPC patients ( n = 4), EV DNA was extracted in agarose plugs by incubation in lysis buffer for 48 h, and high molecular weight DNA was resolved by PFGE. Similar to L-EVs in vitro , patient plasma-derived L-EVs contain high molecular weight DNA (100 kbp–2 Mbp) (indicated by red dashed lines). (d) MYC/PTEN copy number imbalance in PC3 L-EVs was analysed by digital PCR (dPCR) using different amounts of DNA template. (e) MYC/PTEN copy number imbalance was evaluated in L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 6) and compared to MYC/PTEN copy number in normal DNA extracted from the indicated benign cell lines. * p
    Bioanalyzer Dna High Sensitivity Chip Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies bioanalyzer high sensitivity dna 1000 chip
    L-EVs isolated from plasma of mCRPC patients contain large size <t>DNA</t> with tumour aberrations . (a) DNA quantitation in plasma-derived L-EVs and S-EVs, as well as EV-free DNA obtained from mCRPC patients ( n = 4) indicates that a significant portion of circulating DNA is enclosed in L-EVs. The EV-free DNA was extracted from EV-depleted plasma. (b) Chip-based capillary electrophoresis <t>(Bioanalyzer)</t> showing that L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 3) contain high-quality, large size DNA. (c) EVs were isolated from 1 ml of plasma obtained from mCRPC patients ( n = 4), EV DNA was extracted in agarose plugs by incubation in lysis buffer for 48 h, and high molecular weight DNA was resolved by PFGE. Similar to L-EVs in vitro , patient plasma-derived L-EVs contain high molecular weight DNA (100 kbp–2 Mbp) (indicated by red dashed lines). (d) MYC/PTEN copy number imbalance in PC3 L-EVs was analysed by digital PCR (dPCR) using different amounts of DNA template. (e) MYC/PTEN copy number imbalance was evaluated in L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 6) and compared to MYC/PTEN copy number in normal DNA extracted from the indicated benign cell lines. * p
    Bioanalyzer High Sensitivity Dna 1000 Chip, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Small RNA library analysis. ( A – C ) HL small RNA library purification and validation. ( A ) Representative samples of amplified cDNA prepared from HL-RNA were chromatographed on a 5% PAGE gel. The first lane in each panel is a marker composed of three dsDNA fragments of 145, 160, and 500 bp. The region between 145 and 160 bps, corresponding to adapter-ligated constructs derived from miRNA, was excised from the gel ( B ), and purified. The libraries prepared using these purified constructs were validated by analysis with Agilent Bioanalyzer High Sensitivity DNA Chip. ( C ) A representative electropherogram of a purified library. The peak at ~150 bp indicates the presence of cDNA from HL-miRNA. The peaks other than the ones labeled as miRNAs are the lower and upper markers used by the Bioanalyzer system to determine the size and quantity of the library peak. ( D , E ) Length distribution and annotation of small RNA sequencing reads from HL and BT. ( D ) Sequencing reads from all HL or BT miRNA samples were pooled for the purpose of length distribution and annotation analyses. Length distributions by abundance of sequencing reads from BT (clear bars) or HL (filled bars) are shown. Only reads 18–28 nucleotides in size which map uniquely to the UCSC Genome Browser dm6 (NCBI genome/47) Drosophila melanogaster genome are shown. ( E ) Distribution of RNA biotypes represented as the percentages of reads mapping to the indicated small RNAs. Clear and filled bars represent BT and HL samples, respectively. “Other” represents other Drosophila sncRNAs.

    Journal: Genomics Insights

    Article Title: MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner

    doi: 10.4137/GEI.S38147

    Figure Lengend Snippet: Small RNA library analysis. ( A – C ) HL small RNA library purification and validation. ( A ) Representative samples of amplified cDNA prepared from HL-RNA were chromatographed on a 5% PAGE gel. The first lane in each panel is a marker composed of three dsDNA fragments of 145, 160, and 500 bp. The region between 145 and 160 bps, corresponding to adapter-ligated constructs derived from miRNA, was excised from the gel ( B ), and purified. The libraries prepared using these purified constructs were validated by analysis with Agilent Bioanalyzer High Sensitivity DNA Chip. ( C ) A representative electropherogram of a purified library. The peak at ~150 bp indicates the presence of cDNA from HL-miRNA. The peaks other than the ones labeled as miRNAs are the lower and upper markers used by the Bioanalyzer system to determine the size and quantity of the library peak. ( D , E ) Length distribution and annotation of small RNA sequencing reads from HL and BT. ( D ) Sequencing reads from all HL or BT miRNA samples were pooled for the purpose of length distribution and annotation analyses. Length distributions by abundance of sequencing reads from BT (clear bars) or HL (filled bars) are shown. Only reads 18–28 nucleotides in size which map uniquely to the UCSC Genome Browser dm6 (NCBI genome/47) Drosophila melanogaster genome are shown. ( E ) Distribution of RNA biotypes represented as the percentages of reads mapping to the indicated small RNAs. Clear and filled bars represent BT and HL samples, respectively. “Other” represents other Drosophila sncRNAs.

    Article Snippet: These cDNA were excised from the PAGE gel , purified, and analyzed with the Agilent Bioanalyzer High Sensitivity DNA Chip ( ).

    Techniques: Purification, Amplification, Polyacrylamide Gel Electrophoresis, Marker, Construct, Derivative Assay, Chromatin Immunoprecipitation, Labeling, RNA Sequencing Assay, Sequencing

    Standards for MspI digestion and progressive PCR. A) MspI digestion of human genomic DNA isolated from human post-mortem brain tissues. DNA (200 ng) was digested by MspI and run on a 4–20% precast polyacrylamide gel and stained with EtBr. Arrows show three satellite DNA bands characteristic of this enzymatic digestion. B) Agilent 2100 Bioanalyzer chromatogram of MspI digested genomic DNA. C) Bioanalyzer 2100 image of a single library from an MspI digested DNA sample. Notice that the satellite bands (indicated by arrows) are still visible on the Bioanalyzer image. D) Progressive PCR amplification combined with limited PCR extension time allows for size selection and amplification of six bisulfite converted libraries (Lanes 1–5 are distinct RRBS libraries; lane 6 (‘C’) is a negative control). After different progressive PCR cycles (18X, 22X, 24X, or 26X – the same libraries are shown for each cycle number) band intensity increases as cycle number increases. Arrows indicate the three satellite DNA bands that are still visible in these libraries.

    Journal: BMC Genomics

    Article Title: BisQC: an operational pipeline for multiplexed bisulfite sequencing

    doi: 10.1186/1471-2164-15-290

    Figure Lengend Snippet: Standards for MspI digestion and progressive PCR. A) MspI digestion of human genomic DNA isolated from human post-mortem brain tissues. DNA (200 ng) was digested by MspI and run on a 4–20% precast polyacrylamide gel and stained with EtBr. Arrows show three satellite DNA bands characteristic of this enzymatic digestion. B) Agilent 2100 Bioanalyzer chromatogram of MspI digested genomic DNA. C) Bioanalyzer 2100 image of a single library from an MspI digested DNA sample. Notice that the satellite bands (indicated by arrows) are still visible on the Bioanalyzer image. D) Progressive PCR amplification combined with limited PCR extension time allows for size selection and amplification of six bisulfite converted libraries (Lanes 1–5 are distinct RRBS libraries; lane 6 (‘C’) is a negative control). After different progressive PCR cycles (18X, 22X, 24X, or 26X – the same libraries are shown for each cycle number) band intensity increases as cycle number increases. Arrows indicate the three satellite DNA bands that are still visible in these libraries.

    Article Snippet: After purification, 1 μL of the purified library was used for quality control using an Agilent Bioanalyzer 2100 High Sensitivity DNA Chips.

    Techniques: Polymerase Chain Reaction, Isolation, Staining, Amplification, Selection, Negative Control

    DNA methylation levels inversely correlate with expression levels of Esrp2 and Esrp2-as . ( a ) Upper : genomic organization of Esrp2 (dark blue) and Esrp2-as variants 1-4 (v1-4, light blue) and the CGI overlapping the Esrp2 TSS (green). Positions of EPITYPER Amplicons A1-A14 are indicated by horizontal black bars, covering the DMR (pink), the CGI, and CGI shores on both sides. Lower : MCIp-seq detection of methylated DNA fragments in tumors (red) and normal WT mammary glands (blue) of animals at 20 and 24 weeks of age. Each lane represents average reads obtained for three individual samples. ( b , c ) Heatmap of DNA methylation levels in tumor samples ( n =11) and normal mammary gland tissue ( n =9) ( b ), or of various murine cell lines ( c ), with each row representing one individual sample and each column one CpG unit comprising of 1 to 4 individual CpG sites. Methylation levels are depicted by a color-coded gradient from 0% (light yellow) to 100% methylation (blue). Gray squares indicate failed measurements. ( d , e ) Correlation between average methylation of amplicons A1-A14 and Esrp2/Esrp2-as expression levels normalized to three reference genes, calculated by Spearman’s rank correlation for tumor/normal tissues ( d ) and cell lines ( e ). * P

    Journal: Oncogene

    Article Title: Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer

    doi: 10.1038/onc.2017.246

    Figure Lengend Snippet: DNA methylation levels inversely correlate with expression levels of Esrp2 and Esrp2-as . ( a ) Upper : genomic organization of Esrp2 (dark blue) and Esrp2-as variants 1-4 (v1-4, light blue) and the CGI overlapping the Esrp2 TSS (green). Positions of EPITYPER Amplicons A1-A14 are indicated by horizontal black bars, covering the DMR (pink), the CGI, and CGI shores on both sides. Lower : MCIp-seq detection of methylated DNA fragments in tumors (red) and normal WT mammary glands (blue) of animals at 20 and 24 weeks of age. Each lane represents average reads obtained for three individual samples. ( b , c ) Heatmap of DNA methylation levels in tumor samples ( n =11) and normal mammary gland tissue ( n =9) ( b ), or of various murine cell lines ( c ), with each row representing one individual sample and each column one CpG unit comprising of 1 to 4 individual CpG sites. Methylation levels are depicted by a color-coded gradient from 0% (light yellow) to 100% methylation (blue). Gray squares indicate failed measurements. ( d , e ) Correlation between average methylation of amplicons A1-A14 and Esrp2/Esrp2-as expression levels normalized to three reference genes, calculated by Spearman’s rank correlation for tumor/normal tissues ( d ) and cell lines ( e ). * P

    Article Snippet: Size distribution was confirmed on a DNA High sensitivity Chip (Agilent Bioanalyzer), before proceeding with MCIp reaction using a SX8G-V52 robot (Diagenode, Liège, Belgium) for automated processing.

    Techniques: DNA Methylation Assay, Expressing, Methylation

    L-EVs isolated from plasma of mCRPC patients contain large size DNA with tumour aberrations . (a) DNA quantitation in plasma-derived L-EVs and S-EVs, as well as EV-free DNA obtained from mCRPC patients ( n = 4) indicates that a significant portion of circulating DNA is enclosed in L-EVs. The EV-free DNA was extracted from EV-depleted plasma. (b) Chip-based capillary electrophoresis (Bioanalyzer) showing that L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 3) contain high-quality, large size DNA. (c) EVs were isolated from 1 ml of plasma obtained from mCRPC patients ( n = 4), EV DNA was extracted in agarose plugs by incubation in lysis buffer for 48 h, and high molecular weight DNA was resolved by PFGE. Similar to L-EVs in vitro , patient plasma-derived L-EVs contain high molecular weight DNA (100 kbp–2 Mbp) (indicated by red dashed lines). (d) MYC/PTEN copy number imbalance in PC3 L-EVs was analysed by digital PCR (dPCR) using different amounts of DNA template. (e) MYC/PTEN copy number imbalance was evaluated in L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 6) and compared to MYC/PTEN copy number in normal DNA extracted from the indicated benign cell lines. * p

    Journal: Journal of Extracellular Vesicles

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    doi: 10.1080/20013078.2018.1505403

    Figure Lengend Snippet: L-EVs isolated from plasma of mCRPC patients contain large size DNA with tumour aberrations . (a) DNA quantitation in plasma-derived L-EVs and S-EVs, as well as EV-free DNA obtained from mCRPC patients ( n = 4) indicates that a significant portion of circulating DNA is enclosed in L-EVs. The EV-free DNA was extracted from EV-depleted plasma. (b) Chip-based capillary electrophoresis (Bioanalyzer) showing that L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 3) contain high-quality, large size DNA. (c) EVs were isolated from 1 ml of plasma obtained from mCRPC patients ( n = 4), EV DNA was extracted in agarose plugs by incubation in lysis buffer for 48 h, and high molecular weight DNA was resolved by PFGE. Similar to L-EVs in vitro , patient plasma-derived L-EVs contain high molecular weight DNA (100 kbp–2 Mbp) (indicated by red dashed lines). (d) MYC/PTEN copy number imbalance in PC3 L-EVs was analysed by digital PCR (dPCR) using different amounts of DNA template. (e) MYC/PTEN copy number imbalance was evaluated in L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 6) and compared to MYC/PTEN copy number in normal DNA extracted from the indicated benign cell lines. * p

    Article Snippet: DNA quality was assessed using the Bioanalyzer DNA High Sensitivity Chip Kit (Agilent).

    Techniques: Isolation, Quantitation Assay, Derivative Assay, Chromatin Immunoprecipitation, Electrophoresis, Incubation, Lysis, Molecular Weight, In Vitro, Digital PCR

    Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Journal: Journal of Extracellular Vesicles

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    doi: 10.1080/20013078.2018.1505403

    Figure Lengend Snippet: Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Article Snippet: DNA quality was assessed using the Bioanalyzer DNA High Sensitivity Chip Kit (Agilent).

    Techniques: Tunable Resistive Pulse Sensing, Derivative Assay, Quantitation Assay, Purification, Marker, Isolation, HS DSDNA Qubit Assay, Chromatin Immunoprecipitation, Electrophoresis, Lysis